Contents

Module 2

Emails

Sent April 17th:

With apologies for never managing to consolidate these things into one e-mail, I have a few more Module 2 announcements below. Note also that the wiki is currently down. I'll have tomorrow's protocols printed out in some form if the wiki remains inaccessible.

I wanted to put in writing what I only mentioned verbally a long time ago, that is, that you will receive a ***penalty free late day*** if you visit one of the writing fellows to work on your Module 2 report. Recall that walk-in hours are Sundays through Thursdays, 7-9 PM, and that you can also make appointments. https://sites.google.com/site/bewritemit/home

One more tip about taking a holistic view of your data: consider how you might look at Bradford assay and calcium titration data together, particularly if the latter are unusual.

I mentioned a couple of approaches to replicate error analysis to some individuals in T/R section, such as getting a typical difference (should it be absolute or percent?) between replicates and reporting it. Another approach is to perform extra MATLAB runs on your individual normalizations instead of just the average; this approach will give you a sense of how much replicate error impacts error in KD -- at least error from that source. If your replicates are very tight, such a step might be unnecessary.

I look forward to meeting with several of you on Friday... and to starting Module 3 tomorrow!

Sent April 16th:

I wanted to make a few comments as you start working more intensely on your reports:

Please treat what we called the reference mutant, for class purposes, as being of equal importance to your unique design, for report purposes. In fact, I suggest avoiding the terminology "reference mutant" or "positive control." I'll also repeat here the comment from the last homework: "While in a lab context D24H/E67K/T79P/M124S was a known reference, for the purposes of this paper it is a second mutant that you are making a hypothesis about. In other words, you chose to study this mutant because you had a specific hypothesis about it, not simply for historical reasons. If you find this framing a challenge, consider talking with Prof. Jasanoff or your section instructor. (It’s fine to acknowledge that some prior data exists, but that is not the proper justification for the choice you made.)"

Methods:

The basis of the miniprep protocol you used is described here: http://www.ncbi.nlm.nih.gov/pubmed/388356. The method is well known enough that you don’t necessarily need to cite the article, but don’t just cite the 20.109 wiki either. Simply describe the basic principle such that a reader in the field could recgonize the technique that you used.

Note that before IPTG induction, overnight cultures of cells were grown (these reach the saturation/plateau phase) and then sub-cultured to 0.15 OD in the morning and grown to mid-log. A sub-culture was also done prior to making competent cells. For miniprep, on the other hand, overnight cultures were used.

Note that freezing your induced pellets should be considered part of the lysis procedure (that later continues with BPER and other lysis reagents).

Btw, I probably won't have T/R section methods until tomorrow afternoon rather than morning. Stay tuned to the homepage announcements.

Sent April 4th to W/F only:

The OD values for your O/N cultures are on the Day5 Talk page. Hopefully I read Shannon's handwriting okay :)

Note that the really large OD values are *not* typos. Some of the cells took off, perhaps losing the plasmid along the way, and as a result, the protein color got diluted out among all the cells and debris. Shannon thought to spot-check these poor looking pellets by fluorescence microscopy, and it became apparent then that there was indeed some fluorescent protein there.

Sent April 4th to T/R only:

I wanted to assure you that all of the pellets that grew O/N turned greenish/yellowish, even though some stayed at a pretty low OD. Sorry I forgot to post that info on the Talk page! Will do so tomorrow morning.

Sent March 30th:

"For the M2D4 FNT, is the schematic purely for our mutant design? Or is it for the whole module experiment (including mutagenesis, transformation, protein expression, titration, etc.)?"

I thought many of you might be interested in the answer to the question above. We're only asking for a figure showing your mutant design strategy, i.e., for you to define the two mutants in a way that highlights why they might be of interest. We're not interested in seeing the experimental steps even for SDM, much less for all of Module 2. I apologize for any ambiguity in the FNT wording.

Announcements

Module 1

Per requests: Shannon will hold an office hour tonight (Tuesday, March 12) from 9-10 pm in the lab (or the lunchroom across from the lab).

Pre-lab report office hours

Agi's OH will be on Sunday and Monday (3/10 and 3/11) from 2:30-4:30 pm, in 16-336. (I may be taking public transportation in on Sunday, and apologize in advance if I am a little late.)

Shannon's OH will be on Sunday afternoon (just stop by 56-389) and Monday evening 7:30-9:30pm in the lab.

The M1D5 Reagent List has been updated with the exact name of the Qiagen kit. Also, an earlier class email defining the gull samples is now reproduced on the M1D2 Talk page. AgiStachowiak 15:11, 10 March 2013 (EDT)

Shannon will hold an office hour in the lab on Tuesday evening (2/26) from 7:30-8:30pm.

Reminder: Shannon will hold office hours in the lab (56-322) from 11am-12pm today, and Agi will hold office hours in 16-319 from 3:30-4:30 pm. Hope to see some of y'all there!

Wording was corrected in question #1 of the M1D3 FNT, from "gel extraction" to "DNA extraction."

FYI -- here is a link to Marilee's presentation about writing a research paper from Wed, Feb 13. Shannon Hughes 15:03, 15 February 2013 (EST)

Hi folks -- I hope that you all found your figure assignments okay within the M1D3 protocol. I wanted to make clear that the FNT slide is to be done with your partner rather than alone, and that it does not need to be handed in at the beginning of class. You do need to have an electronic copy available during class to revise and ultimately present. AgiStachowiak 16:25, 13 February 2013 (EST)

The first module-based quiz will now be held on M1D3 instead of M1D2, that is, on Thursday and Friday of this coming week. Both M1D1 and M1D2 material will be covered. AgiStachowiak 20:27, 9 February 2013 (EST)